US20110293608A1 - Annexin a2 as immunological target - Google Patents

Annexin a2 as immunological target Download PDF

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US20110293608A1
US20110293608A1 US13/132,509 US200913132509A US2011293608A1 US 20110293608 A1 US20110293608 A1 US 20110293608A1 US 200913132509 A US200913132509 A US 200913132509A US 2011293608 A1 US2011293608 A1 US 2011293608A1
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tumor
annexina2
cell
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Elizabeth Marion Jaffee
Lanqing Huang
Lei Zheng
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Johns Hopkins University
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    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention is related to the area of tumor immunology. In particular, it relates to highly immunogenic proteins found on tumor cells.
  • Pancreatic ductal adenocarcinoma is a devastating malignant disease with a median survival of less than 6 months and an overall 5-year survival rate of 1-4% (Pierantoni, Pagliacci et al. 2008). Lack of early diagnosis and effective systemic treatment are major reasons that account for these dismal survival rates. Morphologic and genetic analyses have implicated pancreatic intraepithelial neoplasm (PanIN) as a precursor lesion of human PDAC.
  • PanIN pancreatic intraepithelial neoplasm
  • PanINs appear to evolve in a stepwise manner through stages (PanIN1A, 1B, 2, 3) that display increasing cellular atypia and accumulate clonal mutations or aberrant expression of oncogenes or tumor suppressor genes such as K-Ras, p 16, p 53, and DPC4/SMAD4 in the course of progression to PDAC (Goggins, Kern et al. 1999).
  • drugs that target these molecular abnormalities have not yet translated into improved clinical responses (Strimpakos, Saif et al. 2008).
  • the aggressive nature of PDAC is featured by its early invasion and metastasis.
  • Cancer immunotherapy is an emerging approach for the treatment of pancreatic cancer.
  • Early clinical studies are also providing critical human reagents for developing methods to identify new candidate proteins and biologic pathways.
  • immunized lymphocytes and sera are being used to develop functional genomic and proteomic approaches for identifying those proteins that are relevant to the cancer.
  • We have developed an allogeneic, GM-CSF secreting pancreatic cancer vaccine approach (Jaffee, Hruban et al. 2001).
  • Phase I and II trials evaluating this vaccine in patients with resected PDAC have demonstrated both clinical and immunologic responses (Jaffee, Hruban et al. 2001; Laheru, Lutz et al. 2008) (Lutz et al. Manuscript submitted).
  • Humoral immune response is an important and integrated part of the immune mechanisms by which a host defends itself against pathogen assault.
  • Antibodies generated from vaccinations are a major factor that has protected generations of children from deadly infectious diseases.
  • antibodies can also mediate pathogenesis in many autoimmune diseases in which the antibodies target cellular components of the host, i.e., auto-antigens, which under normal physiological conditions are tolerated by host immune system (1).
  • auto-antigens i.e., antigens in cancer come from within. Indeed, a common repertoire of autoantibodies was found to be shared by cancer and autoimmune disease patients (2). In addition, a majority of these autoantibodies are directed against intracellular components, which leads to the assumption that autoantigens in both cancer and autoimmune diseases emerge from damaged cells (3).
  • autoantibodies can be detected at the very early pre-malignant stage of cancer development when there is no obvious cancer cell death or inflammation (4). It appears that aberrant gene expression, post-translational modification, and/or protein re-localization in cancer cells gives rise to antigens that are expressed either (a) to a greater extent than in normal cells or (b) as “altered” molecules absent in corresponding normal cells, or (c) in cell compartments where they are not supposed to be under normal conditions (ectopic expression), e.g., nuclear or cytoplasmic proteins appearing on the cell surface and non-secreted proteins being secreted to extracellular milieu. These molecules are collectively named tumor associated antigens (TAAs).
  • TAAs tumor associated antigens
  • ganglioside-reactive antibodies detected in cancer patients were of the IgM class; this was also the immunoglobulin class induced by vaccination of melanoma patients with pure or modified gangliosides (10, 11), which means that no memory response was developed. Nonetheless, ganglioside GM2 antibody production in melanoma patients was associated with a prolonged disease-free interval and survival (12).
  • serological identification of antigens by recombinant expression cloning (SEREX), serological proteome analysis (SERPA) and protein microarrays a large number of antibody-reactive tumor associated antigens (TAAs) were identified.
  • TAAs tumor-associated antitumors
  • pancreatic cancer in which TAAs are poorly characterized, whole tumor cell vaccines present a good source for investigating TAAs immunotherapy.
  • Vaccination with irradiated whole tumor cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce potent systemic immune responses that are capable of eradicating tumors (24).
  • GM-CSF-secreting tumor vaccines can induce both CD4 + and CD8 + T cell-mediated antitumor responses and a broad range of antibody responses (25-27).
  • a pharmaceutical composition comprising monoclonal antibodies.
  • the antibodies specifically bind to AnnexinA2.
  • An aspect of the invention is a method for treating a patient with a tumor to diminish risk of tumor invasion and/or metastatic progression.
  • Monoclonal antibodies which specifically bind to AnnexinA2 are administered to the patient.
  • Another aspect of the invention is a method of treating a patient with a tumor to diminish risk of tumor invasion and/or metastatic progression.
  • ANXA2 protein or a nucleic acid encoding ANXA2 to the patient is administered to the patient.
  • a T cell or B cell response to ANXA2 is thereby induced.
  • Still another aspect of the invention is a method of screening for candidate drugs which inhibit tumor invasion and/or metastasis.
  • One or more test substances are contacted with pancreatic cancer cells in culture. Subcellular localization of ANXA2 after the contacting is determined.
  • One or more test substances are identified as candidate drugs for inhibiting tumor invasion and/or metastasis if the one or more test substances inhibit translocation of ANXA2 to the cell surface.
  • a method of screening for candidate drugs which inhibit tumor invasion and/or metastasis is provided.
  • One or more test substances is contacted with pancreatic cancer cells in culture.
  • Phosphorylation status of Tyr23 of ANXA2 is determined.
  • One or more test substances are identified as candidate drugs for inhibiting tumor invasion and/or metastasis if the one or more test substances inhibit phosphorylation of Tyr23 of ANXA2.
  • Yet another aspect of the invention is a method of screening for candidate drugs which inhibit tumor invasion and/or metastasis.
  • One or more test substances is contacted with cancer cells in an animal model of pancreatic cancer. Subcellular localization of ANXA2 after the contacting is determined. The one or more test substances is identified as a candidate drug for inhibiting tumor invasion and/or metastasis if the one or more test substances inhibit translocation of ANXA2 to the cell surface.
  • a further aspect of the invention is a method of screening for candidate drugs which inhibit tumor invasion and/or metastasis.
  • One or more test substances is contacted with cancer cells in an animal model of pancreatic cancer. Phosphorylation status of Tyr23 of ANXA2 is determined.
  • the one or more test substances are identified as candidate drugs for inhibiting tumor invasion and/or metastasis if the one or more test substances inhibit phosphorylation of Tyr23 of ANXA2.
  • Another embodiment of the invention is a vaccine for treating patients with a tumor which has been resected.
  • the vaccine comprises ANXA2 or a nucleic acid encoding ANXA2.
  • FIG. 1 Chronological changes of vaccine-specific antibody response in four patients.
  • Total cell lysate from two vaccine tumor lines was resolved at 40 ⁇ g proteins/lane on 4-12% Bis-Tris SDS-PAGE and immunoblotted with 1:1,000 dilutions of serum samples from vaccinated patients 9, 12, 27, and 53 who survived greater than 5 years post-pancreaticoduodenectomy.
  • Specific antibodies in the serum were detected with peroxidase-conjugated goat anti-human IgG. Date of serum sample collection is shown on top of each lane. Vaccination is indicated with an arrow.
  • FIGS. 2A-2D Vaccine-specific antibody response in all 60 vaccinated patients and 22 healthy donors. Total cell lysate from two vaccine tumor lines was resolved at 40 ⁇ g proteins/lane on 4-12% Bis-Tris SDS-PAGE and immunoblotted with 1:1,000 dilutions of pre-vaccination (left lane) versus post-3 rd vaccination (right lane) serum samples from each patient who completed at least 3 vaccinations. For patients who completed only the 1 st vaccination and the healthy donors, only pre-vaccination (patients) or a single time point sera (donors) were tested. Specific antibodies in the serum were detected with peroxidase-conjugated goat anti-human IgG. ( FIG.
  • FIGS. 5A-5B Antibody response to AnnexinA2, enolase, RhoGDI ⁇ or HSP60 was enhanced by radiation and chemotherapy. Serum samples were tested by ELISA for antibody response against recombinant proteins AnnexinA2, enolase, RhoGDI, or HSP60. OD 450 values at a serum dilution of 1:400 are shown.
  • FIG. 5A Pre-existing antibody titer in pre-vaccination serum samples from vaccinated patients and serum samples from healthy donors (left panel) and the time course of antibody titer changes in patients who completed at least 3 vaccinations (right panel). Each symbol represents a patient or donor.
  • Antibody titers shown are at pre-vaccination (Pre-Vac), 14 days post-1 st vaccination (Vac 1), 1 month post-radiochemotherapy (Post-Rx), and 1 month post-3 rd vaccination (Vac 3).
  • a solid symbol indicates an elevated antibody titer in the post-vaccination sera.
  • FIG. 5 (B) The detailed time course of antibody titer changes at all time points in patients 9, 10, and 47 who had an elevated antibody titer post-radiochemotherapy and patient 6 whose antibody titer to enolase declined over time.
  • FIGS. 6A-6C Intact antibody response to influenza proteins in vaccinated patients. Serum samples were tested by ELISA for antibody response against recombinant influenza fusion protein NPM1. OD 450 values at a serum dilution of 1:400 are shown.
  • FIG. 6 (A) Pre-existing anti-NPM1 antibody in pre-vaccination serum samples from vaccinated patients and serum samples from healthy donors. Each symbol represents a patient or donor.
  • FIG. 6B The time course of anti-NPM1 antibody titer changes in patients who completed at least 3 vaccinations.
  • FIG. 7A-7E show purified recombinant His6-tagged AnnexinA2 (His6-ANXA2) on a SDS-PAGE gel stained with commassie blue.
  • FIG. 7B Purified His6 tagged AnnexinA2 (ANXA2) on a SDS-PAGE gel was western-blotted by pre- and post-vaccination serum. Patients marked by * had antibody induction, which was manifested by stronger signals of ANXA2 in western blot with post-vaccination serum vs. pre-vaccination serum.
  • FIGS. 7C-7E immunohistochemistry staining of AnnexinA2 in human pancreatic adenocarcinoma. AnnexinA2 expression with score 0, 1, 2, 3 was indicated. PanINs and PDAC were indicated.
  • FIGS. 9A-9C In vitro invasion of multiple pancreatic cancer cell lines. Invaded cells were measured by MTT assays. Shown are average MTT units on three parallel experiments after they were normalized by total cell numbers.
  • FIG. 9B expression of ANXA2 in each cell lines demonstrated by immunoblot analyses with anti-ANXA2 polyclonal antibody. Lanes 1-12 correspond to human pancreatic cancer cell lines: Panc01.28, Panc10.5, Panc2.8, Panc2.03, Panc4.03, PancTS0129, Panc3.11, Panc2.13, Panc6.03, Panc9.3.96, and Panc2.43, respectively; lane 13, human pancreatic para-cancerous fibroblast cells.) FIG.
  • fluorescent immunostaining showed predominant cell surface localization of ANXA2 in representative cells with higher invasion capacity (Panc10.05, Panc2.43, Panc2.03), but not in cells with lower invasion capacity (human fibroblast, nuclear/cytoplasmic staining; Panc3.11, perinuclear staining; MiaPaca-2, cytoplasmic/nuclear staining).
  • FITC indicates the images of immunostaining with rabbit anti-ANXA2 polyclonal antibody and FITC-conjugated secondary antibody.
  • FITC+DAPI indicates the overlapped images of FITC staining of ANXA2 and DAPI staining of nuclei.
  • FIGS. 10A-10C show that FIG. 10A .
  • Panc10.05 and Panc3.11 cells were either incubated with the EGTA containing buffer or the EGTA-free buffer.
  • the two different elutions from two different cell lines as indicated were immunoprecipitated by anti-ANXA2 antibodies (lanes 1-4) or anti-phosphotyrosine antibodies (lanes 9-12).
  • the two cell lines were lysed and the lysates were immunoprecipated by the anti-phosphotyrosine antibodies (anti-pTry) (lanes 5-8).
  • FIG. 10 B shows
  • GFP-tagged wild-type ANXA2 a, GFP-tagged wild-type ANXA2; b, GFP-tagged Y23A-mutated ANXA2; c, GFP-tagged Y23E-mutated ANXA2.
  • FIGS. 11A-11B FIG. 11A . FLAG-tagged ANXA2 expression in Panc10.05 cells transfected by the pcDNA-based plasmid vector alone (lanes 1,5,9,13), the plasmid carrying ANXA2 WT -FLAG (lanes 2,6,10,14), the plasmid carrying ANXA2 Y23A -FLAG (lanes 3,7,11,15), or the plasmid carrying ANXA2 Y23E -FLAG (lanes 4,8,12,16).
  • FIG. 12 Quantitative real-time PCR analyses of E-cadherin, slug, and vimentin mRNA expression in a pair of Panc 10.05 cell lines, one with and the other without knockdown of ANXA2 by siRNA. The relative ratios of mRNA expression with TGF ⁇ 1 treatment vs. without TGF treatment are shown. The data were normalized with ⁇ -actin expression.
  • AnnexinA2 induces a strong immunological response in patients vaccinated with a whole cell tumor vaccine. Moreover, the inventors have found that in cancer cells, AnnexinA2 translocates to the cell surface and is phosphorylated on Tyrosine 23. Both the phosphorylation and the translocation are critical for tumor cell invasiveness and metastasis. An antibody response to AnnexinA2 in vaccinated patients correlates with a favorable clinical response, i.e., an improvement in length of disease-free survival. These results identify AnnexinA2 as an excellent immune target for inhibition of invasiveness and metastasis.
  • Immunological targeting of AnnexinA2 can be accomplished either passively, by administration of antibodies that specifically bind to AnnexinA2, or actively, by vaccination with peptide or nucleic acid vaccines.
  • the peptide or nucleic acid vaccines comprise or encode, respectively, at least one T or B cell epitope of AnnexinA2.
  • Antibodies according to the invention may be monoclonal or polyclonal. They may be human, mouse, rat, goat, horse, or chimeric. They may be humanized antibodies. Such generic types of antibodies and means of making them are well known in the art.
  • Nucleic acids may be administered as part of vectors, for example viral or plasmid vectors. Naked DNA or protein-complexed DNA or polymer-complexed DNA, or viral encapsidated DNA may be used. Similarly RNA can be used with appropriate systems, such as RNA viruses. Protein vaccines may additionally comprise adjuvants.
  • the vaccines of the present invention can be administered by any means known in the art for inducing a T cell cytolytic response or a B cell humoral response. These means include oral administration, intravenous injection, percutaneous scarification, subcutaneous injection, intramuscular injection, and intranasal administration.
  • the vaccines can be administered intradermally by gene gun. Gold particles coated with DNA may be used in the gene gun. Other inoculation routes as are known in the art can be used.
  • agents which are beneficial to raising a cytolytic T cell response may be used as well.
  • agents are termed herein carriers. These include, without limitation, B7 costimulatory molecule, interleukin-2, interferon- ⁇ , GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligand, CD40/CD40 ligand, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions.
  • BCG Bacille Calmette-Guerin
  • Carriers for inducing a T cell immune response which preferentially stimulate a cytolytic T cell response versus an antibody response are preferred, although those that stimulate both types of response can be used as well.
  • the agent is a polypeptide
  • the polypeptide itself or a polynucleotide encoding the polypeptide can be administered.
  • the carrier can be a cell, such as an antigen presenting cell (APC) or a dendritic cell.
  • APC antigen presenting cell
  • Antigen presenting cells include such cell types aas macrophages, dendritic cells and B cells.
  • Other professional antigen-presenting cells include monocytes, marginal zone Kupffer cells, microglia, Langerhans' cells, interdigitating dendritic cells, follicular dendritic cells, and T cells. Facultative antigen-presenting cells can also be used. Examples of facultative antigen-presenting cells include astrocytes, follicular cells, endothelium and fibroblasts.
  • the carrier can be a bacterial cell that is transformed to express the polypeptide or to deliver a polynucleoteide which is subsequently expressed in cells of the vaccinated individual.
  • Adjuvants such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response.
  • adjuvants include the synthetic adjuvant QS-21 comprising a homogeneous saponin purified from the bark of Quillaja saponaria and Corynebacterium parvum (McCune et al., Cancer, 1979; 43:1619). It will be understood that the adjuvant is subject to optimization. In other words, the skilled artisan can engage in routine experimentation to determine the best adjuvant to use.
  • Preservatives such as thimerosal or 2-phenoxy ethanol
  • Preservatives can be added to slow or stop the growth of bacteria or fungi resulting from inadvertent contamination, especially as might occur with vaccine vials intended for multiple uses or doses.
  • Stabilizers such as lactose or monosodium glutamate (MSG), can be added to stabilize the vaccine formulation against a variety of conditions, such as temperature variations or a freeze-drying process.
  • Viral vectors can be used to administer polynucleotides encoding a polypeptide comprising an AnnexinA2 epitope.
  • Such viral vectors include vaccinia virus and avian viruses, such as Newcastle disease virus. Others may be used as are known in the art.
  • One particular method for administering polypeptide vaccine is by pulsing the polypeptide onto an APC or dendritic cell in vitro.
  • the polypeptide binds to MHC molecules on the surface of the APC or dendritic cell.
  • Prior treatment of the APCs or dendritic cells with interferon- ⁇ can be used to increase the number of MHC molecules on the APCs or dendritic cells.
  • the pulsed cells can then be administered as a carrier for the polypeptide.
  • Peptide pulsing is taught in Melero et al., Gene Therapy 7:1167 (2000).
  • Naked DNA can be injected directly into the host to produce an immune response.
  • Such naked DNA vaccines may be injected intramuscularly into human muscle tissue, or through transdermal or intradermal delivery of the vaccine DNA, typically using biolistic-mediate gene transfer (i.e., gene gun).
  • biolistic-mediate gene transfer i.e., gene gun.
  • Some reviews describing the gene gun and muscle injection delivery strategies for DNA immunization include Tuting, CUM Opin. Mol. Ther. (1999) 1: 216-25, Robinson, Int. J. Mol. Med. (1999) 4: 549-55, and Mumper and Ledbur, Mol. Biotechnol. (2001) 19: 79-95.
  • Other possible methods for delivering plasmid DNA includes electroporation and iontophoreses.
  • Another possible gene delivery system comprises ionic complexes formed between DNA and polycationic liposomes (see, e.g., Caplen et al. (1995) Nature Med. 1: 39). Held together by electrostatic interaction, these complexes may dissociate because of the charge screening effect of the polyelectrolytes in the biological fluid.
  • a strongly basic lipid composition can stabilize the complex, but such lipids may be cytotoxic.
  • intracellular and intercellular targeting strategies may further enhance the AnnexinA2-specific antitumor effect.
  • intracellular targeting strategies and intercellular spreading strategies have been used to enhance MHC class I or MHC class II presentation of antigen, resulting in potent CD8+ or CD 4+T cell-mediated antitumor immunity, respectively.
  • MHC class I presentation of a model antigen, HPV- 16 E7 was enhanced using linkage of Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen, et al., (2000), Cancer Research, 60: 1035-1042), calreticulin (Cheng, et al., (2001) J Clin Invest, 108:669-678) or the translocation domain (domain II) of Pseudomonas aeruginosa exotoxin A (ETA (dII)) (Hung, et al., (2001) Cancer Research, 61: 3698-3703) to E7 in the context of a DNA vaccine.
  • HSP70 Mycobacterium tuberculosis heat shock protein 70
  • calreticulin Choeng, et al., (2001) J Clin Invest, 108:669-678
  • ETA (dII) the translocation domain of Pseudomonas aeruginosa exotoxi
  • the sorting signals of the lysosome associated membrane protein (LAMP-1) have been linked to the E7 antigen, creating the Sig/E7/LAMP-1 chimera (Ji, et al, (1999), Human Gene Therapy, 10: 2727-2740).
  • LAMP-1 lysosome associated membrane protein
  • an intercellular strategy that facilitates the spread of antigen between cells can be used. This improves the potency of DNA vaccines as has been shown using herpes simplex virus (HSV-1) VP22, an HSV-1 tegument protein that has demonstrated the remarkable property of intercellular transport and is capable of distributing protein to many surrounding cells (Elliot, et al., (1997) Cell, 88: 223-233).
  • HSV-1 VP22 herpes simplex virus
  • HSV-1 tegument protein that has demonstrated the remarkable property of intercellular transport and is capable of distributing protein to many surrounding cells.
  • Such enhanced intercellular spreading of linked protein results in enhancement of antigen-specific CD8+ T
  • Polypeptides for immunization to raise a cytolytic T cell response are optionally from 8 to 25 amino acid residues in length. Any 8 contiguous amino acids of AnnexinA2 can be used as well.
  • the polypeptides can be fused to other such epitopic polypeptides, or they can be fused to carriers, such as B-7, interleukin-2, or interferon- ⁇ .
  • the fusion polypeptide can be made by recombinant production or by chemical linkage, e.g., using heterobifunctional linking reagents. Mixtures of polypeptides can be used. These can be mixtures of epitopes for a single allelic type of an MHC molecule, or mixtures of epitopes for a variety of allelic types.
  • the polypeptides can also contain a repeated series of an epitope sequence or different epitope sequences in a series.
  • Nucleic acids encoding AnnexinA2 may be used in any form, including as cDNA, genomic, full-length coding sequence, partial coding sequence, full-length transcript or copy of it, or short fragments encoding one or more epitopes. Sequence of AnnexinA2 nucleic acids are known in the art, and any can be used including NM — 001002858.2, NM — 001136015.2, NM — 004039.2, and NM — 001002857.1. Any of these or any which encode the protein products, such as NP — 001002858.1 and NP — 001002857.1 may be used.
  • Plasmids and viral vectors can be used to express a tumor antigen protein in a host cell.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a nucleotide sequence derived from the cloning of AnnexinA2 proteins, encoding all or a selected portion of the full-length protein can be used to produce a recombinant form of an AnnexinA2 polypeptide via microbial or eukaryotic cellular processes.
  • the coding sequence can be ligated into a vector and the loaded vector can be used to transform or transfect hosts, either eukaryotic (e.g., yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells.
  • eukaryotic e.g., yeast, avian, insect or mammalian
  • prokaryotic bacterial
  • expression vectors used for expressing a polypeptide in vivo or in vitro contain a nucleic acid encoding an antigen polypeptide, operably linked to at least one transcriptional regulatory sequence. Regulatory sequences are art-recognized and can be selected to direct expression of the subject proteins in the desired fashion (time and/or place). Transcriptional regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • Suitable vectors for the expression of a polypeptide comprising HLA-binding epitopes include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli .
  • Mammalian expression vectors may contain both prokaryotic and eukaryotic sequences in order to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that can be expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and selection in both prokaryotic and eukaryotic cells.
  • viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • Vaccinia and avian virus vectors can also be used.
  • the methods which may be employed in the preparation of vectors and transformation of host organisms are well known in the art.
  • suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.
  • a polypeptide described herein, or a polynucleotide encoding the polypeptide is delivered to a host organism in an immunogenic composition comprising yeast.
  • an immunogenic composition comprising yeast.
  • live yeast DNA vaccine vectors for antigen delivery has been reviewed and reported to be efficacious in a mouse model using whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens (see Stubbs et al. (2001) Nature Medicine 7: 625-29).
  • yeast vaccine vectors are known in the art. Furthermore, U.S. Pat. No. 5,830,463, the contents of which are incorporated herein by reference, describes particularly useful vectors and systems which can be used with the instant invention.
  • the use of yeast delivery systems may be particularly effective for use in the tumor/cancer vaccine methods and formulations, as yeast appears to trigger cell-mediated immunity without the need for an additional adjuvant.
  • Particularly preferred yeast vaccine delivery systems are nonpathogenic yeast carrying at least one recombinant expression system capable of modulating an immune response.
  • Bacteria can also be used as carriers for the epitopes of the present invention. Typically the bacteria used are mutant or recombinant.
  • the bacterium is optionally attenuated. For instance, a number of bacterial species have been developed for use as vaccines and can be used in the present invention, including, but not limited to, Shigella flexneri, E. coli, Listeria monocytogenes, Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi or mycobacterium.
  • the bacterial vector used in the immunogenic composition may be a facultative, intracellular bacterial vector.
  • the bacterium may be used to deliver a polypeptide described herein to antigen-presenting cells in the host organism.
  • Bacterially mediated gene transfer is particularly useful in genetic vaccination by intramuscular, intradermal, or oral administration of plasmids; such vaccination leads to antigen expression in the vaccinee.
  • bacteria can provide adjuvant effects and the ability to target inductive sites of the immune system.
  • bacterial vaccine vectors have almost unlimited coding capacity.
  • the use of bacterial carriers is often associated with still other significant benefits, such as the possibility of direct mucosal or oral delivery.
  • Other direct mucosal delivery systems (besides live viral or bacterial vaccine carriers) which can be used include mucosal adjuvants, viral particles, ISCOMs, liposomes, and microparticles.
  • Attenuated mucosal pathogens which may be used in the invention include: L. monocytogenes, Salmonella spp., V. cholorae, Shigella spp., mycobacterium, Y enterocolitica , and B. anthracis .
  • Commensal strains which can be used in the invention include: S. gordonii, Lactobacillus spp., and Staphylococcus spp.
  • the genetic background of the carrier strain used in the formulation, the type of mutation selected to achieve attenuation, and the intrinsic properties of the immunogen can be adjusted to optimize the extent and quality of the immune response elicited.
  • the general factors to be considered to optimize the immune response stimulated by the bacterial carrier include: selection of the carrier; the specific background strain, the attenuating mutation and the level of attenuation; the stabilization of the attenuated phenotype and the establishment of the optimal dosage.
  • Other antigen-related factors to consider include: intrinsic properties of the antigen; the expression system, antigen-display faun and stabilization of the recombinant phenotype; co-expression of modulating molecules and vaccination schedules.
  • Salmonella typhimurium can be used as a bacterial vector in the immunogenic compositions of the invention. Use of this bacterium as an effective vector for a vaccine has been demonstrated in the art. For instance, the use of S. typhimurium as an attenuated vector for oral somatic transgene vaccination has been described (see Darji et al. (1997) Cell 91: 765-775; and Darji et al. (2000) FEMS Immun and Medical Microbiology 27: 341-9). Indeed most knowledge of bacteria-mediated gene transfer has been acquired using attenuated S. typhimurium as carrier. Two metabolically attenuated strains that have been used include S. typhimurium aroA, which is unable to synthesize aromatic amino acids, and S.
  • listeriolysin and actA two virulence factors of L. monocytogenes
  • beta-galactosidase ⁇ -gal
  • Cytotoxic and helper T cells as well as specific antibodies could be detected against these antigens following oral application of a single dose of the recombinant salmonella .
  • immunization with Salmonella carrying a listeriolysin-encoding expression plasmid elicited a protective response against a lethal challenge with L. monocytogenes .
  • Oral transgene vaccination methodology has now been extended to include protective responses in herpes simplex virus 2 and hepatitis B infection models, with cell-mediated immune responses detected at the mucosal level.
  • Salmonella can be used to induce a tumor growth retarding response against the murine melanoma B16; the Salmonella carry minigenes encoding epitopes of the autologous tumor antigens gp100 and TRP2 fused to ubiquitin. This suggests that under such circumstances peripheral tolerance towards autologous antigens can be overcome. This was confirmed by the same group (Lode et al. (2000) Med Ped Oncol 35: 641-646) using similar constructs of epitopes of tyrosine hydroxylase as autologous antigen in a murine neuroblastoma system.
  • Salmonella typhi Another bacterial vector which may be used in the immunogenic compositions described herein is Salmonella typhi .
  • improved strains include those attenuated by a mutation in guaBA, which encodes an essential enzyme of the guanine biosynthesis pathway (Pasetti et al., Infect. Immun. (2002) 70:4009-18; Wang et al., Infect. Immun. (2001) 69:4734-41; Pasetti et al., Clin. Immunol. (1999) 92:76-89).
  • Additional references describing the use of Salmonella typhi and/or other Salmonella strains as delivery vectors for DNA vaccines include the following: Lundin, Infect. Immun. (2002) 70:5622-7; Devico et al., Vaccine, (2002) 20:1968-74; Weiss et al., Biol. Chem. (2001) 382:533-41; and Bumann et al., FEMS Immunol. Med. Microbiol. (2000) 27:357-64.
  • the vaccines and immunogenic compositions of the present invention can employ Shigella flexneri as a delivery vehicle.
  • S. flexneri represents the prototype of a bacterial DNA transfer vehicle as it escapes from the vacuole into the cytosol of the host cell.
  • Several attenuated mutants of S. flexneri have been used successfully to transfer DNA to cell lines in vitro.
  • Auxotrophic strains were defective in cell-wall synthesis (Sizemore et al. (1995) Science 270: 299-302 and Courvalin et al. (1995) C R Acad Sci Ser III, 318: 1207-12), synthesis of aromatic amino acids (Powell et al. (1996) Vaccines 96: Molecular Approaches to the Control of Infectious Disease; Cold Spring Harbor Laboratory Press) or synthesis of guanine nucleotides (Anderson et al. (2000) Vaccine 18: 2193-2202).
  • the vaccines and immunogenic compositions of the present invention may comprise Listeria monocytogenes (Portnoy et al, Journal of Cell Biology, 158:409-414 (2002); Glomski et al., Journal of Cell Biology, 156:1029-1038 (2002)).
  • L. monocytogenes The ability of L. monocytogenes to serve as a vaccine vector has been reviewed in Wesikirch, et al., Immunol. Rev. 158:159-169 (1997).
  • Strains of Listeria monocytogenes have recently been developed as effective intracellular delivery vehicles of heterologous proteins providing delivery of antigens to the immune system to induce an immune response to clinical conditions that do not permit injection of the disease-causing agent, such as cancer (U.S. Pat. No.
  • L. monocytogenes vaccine expressing an lymphocytic choriomeningitis virus (LCMV) antigen has also been shown to induce protective cell-mediated immunity to the antigen (Shen et al., Proc. Natl. Acad. Sci. USA, 92: 3987-3991 (1995)).
  • LCMV lymphocytic choriomeningitis virus
  • L. monocytogenes As a facultative intracellular bacterium, L. monocytogenes elicits both humoral and cell-mediated immune responses. Following entry of Listeria into a cell of the host organism, the Listeria produces Listeria -specific proteins that enable it to escape from the phagolysosome of the engulfing host cell into the cytosol of that cell.
  • L. monocytogenes proliferates, expressing proteins necessary for survival, but also expressing heterologous genes operably linked to Listeria promoters. Presentation of peptides of these heterologous proteins on the surface of the engulfing cell by MHC proteins permit the development of a T cell response.
  • Two integration vectors that are useful for introducing heterologous genes into the bacteria for use as vaccines include pL1 and pL2 as described in Lauer et al., Journal of Bacteriology, 184: 4177-4186 (2002).
  • L. monocytogenes useful in immunogenic compositions.
  • the ActA protein of L. monocytogenes is sufficient to promote the actin recruitment and polymerization events responsible for intracellular movement.
  • a human safety study has reported that oral administration of an actA/plcB-deleted attenuated form of Listeria monocytogenes caused no serious sequelae in adults (Angelakopoulos et al., Infection and Immunity, 70:3592-3601 (2002)).
  • Other types of attenuated forms of L. monocytogenes have also been described (see, for example, WO 99/25376 and U.S. Pat. No. 6,099,848, which describe auxotrophic, attenuated strains of Listeria that express heterologous antigens).
  • Yersinia enterocolitica is another intraceullular bacteria that can optionally be used as a bacterial vector in immunogenic compositions of the present invention.
  • the use of attenuated strains of Yersini enterocolitica as vaccine vectors is described in PCT Publication WO 02/077249.
  • the immunogenic compositions of the invention comprise mycobacterium, such as Bacillus Calmette-Guerin (BCG).
  • BCG Bacillus Calmette-Guerin
  • the Bacillus of Calmette and Guerin has been used as a vaccine vector in mouse models (Gicquel et al., Dev. Biol. Stand 82:171-8 (1994)). See also, Stover et al., Nature 351: 456-460 (1991).
  • viral vectors can be used.
  • the viral vector will typically comprise a highly attenuated, non-replicative virus.
  • Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, herpes simplex virus, avian viruses, such as Newcastle disease virus, poxviruses such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including, but not limited to, the retroviral vectors.
  • Vaccinia vectors and methods useful in immunization protocols are described in U.S. Pat. No. 4,722,848.
  • Retroviral vectors include murine leukemia virus, and lentiviruses such as human immunodeficiency virus.
  • Replication-defective retroviral vectors harboring a polynucleotide of the invention as part of the retroviral genome can be used. Such vectors have been described in detail. (Miller, et al. (1990) Mol. Cell. Biol. 10:4239; Kolberg, R. (1992) J. NIH Res. 4:43; Cornetta, et al. (1991) Hum. Gene Therapy 2:215).
  • Adenovirus and adeno-associated virus vectors useful in this invention may be produced according to methods already taught in the art. (See, e.g., Karlsson, et al. (1986) EMBO 5:2377; Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzcyzka (1992) Current Top. Microbiol. Immunol. 158:97-129; Gene Targeting: A Practical Approach (1992) ed. A. L. Joyner, Oxford University Press, NY). Several different approaches are feasible.
  • Alpha virus vectors such as Venezuelan Equine Encephalitis (VEE) virus, Semliki Forest virus (SFV) and Sindbis virus vectors, can be used for efficient gene delivery. Replication-deficient vectors are available. Such vectors can be administered through any of a variety of means known in the art, such as, for example, intranasally or intratumorally. See Lundstrom, Curr. Gene Ther. 2001 1:19-29.
  • Additional references describing viral vectors which could be used in the methods of the present invention include the following: Horwitz, M. S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F. et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller, et al.
  • DNA is complexed with liposomes or ligands that often target cell surface receptors.
  • the complex is useful in that it helps protect DNA from degradation and helps target plasmid to specific tissues.
  • the complexes are typically injected intravenously or intramuscularly.
  • Polynucleotides used as vaccines can be used in a complex with a colloidal dispersion system.
  • a colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • One colloidal system that may be used with this invention is a lipid-complexed or liposome-formulated DNA.
  • a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5′ untranslated region and elimination of unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995).
  • Formulation of DNA, e.g., with various lipid or liposome materials may then be effected using known methods and materials and delivered to the recipient mammal.
  • complex coacervation is a process of spontaneous phase separation that occurs when two oppositely charged polyelectrolytes are mixed in an aqueous solution.
  • the electrostatic interaction between the two species of macromolecules results in the separation of a coacervate (polymer-rich phase) from the supernatant (polymer-poor phase).
  • This phenomenon can be used to form microspheres and encapsulate a variety of compounds.
  • the encapsulation process can be performed entirely in aqueous solution and at low temperatures, and has a good chance, therefore, of preserving the bioactivity of the encapsulant.
  • the candidate vaccine containing the desired tumor antigen can be administered to a population of mice either before or after challenge with a tumor cell line.
  • the mouse model can be used to test for both therapeutic and prophylactic effects.
  • Vaccination with a candidate vaccine can be compared to control populations that are either not vaccinated, vaccinated with vehicle alone, or vaccinated with a vaccine that expresses an irrelevant antigen. If the vaccine is a recombinant microbe, its relative efficacy can be compared to a population of microbes in which the genome has not been modified to express the antigen.
  • the effectiveness of candidate vaccine can be evaluated in terms of effect on tumor or ascites volume or in terms of survival rates.
  • the tumor or ascites volume in mice vaccinated with candidate vaccine may be about 5%, about 10%, about 25%, about 50%, about 75%, about 90% or about 100% less than the tumor volume in mice that are either not vaccinated or are vaccinated with vehicle or a vaccine that expresses an irrelevant antigen.
  • the differential in tumor or ascites volume may be observed at least about 10, at least about 17, or at least about 24 days following the implantation of the tumor cells into the mice.
  • the median survival time in mice vaccinated with a nucleic acid-modified microbe may be, for example, at least about 2, at least about 5, at least about 7, or at least about 10 days longer than in mice that are either not vaccinated or are vaccinated with vehicle or a vaccine that expresses an irrelevant antigen.
  • the mouse model can be used to test any kind of cancer treatment known in the art.
  • the candidate cancer treatment may be radiation therapy, chemotherapy, or surgery.
  • the candidate cancer treatment may be a combination of two or more therapies or prophylaxes, including but not limited to anti-cancer agents, anti-tumor vaccines, radiation therapy, chemotherapies, and surgery.
  • any oncogene known in the art can be used to make the peritoneal or mesothelium cell line for making a mouse model.
  • oncogenes include without limitation, Ki-ras, Erb-B2, N-ras, N-myc, L-myc, C-myc, ABL1, EGFR, Fos, Jun, c-Ha-ras, and SRC.
  • the vaccines, polynucleotides, polypeptides, cells, and viruses of the present invention can be administered to either human or other mammals.
  • the other mammals can be domestic animals, such as goats, pigs, cows, horses, and sheep, or can be pets, such as dogs, rabbits, and cats.
  • the other mammals can optionally be experimental subjects, such as mice, rats, rabbits, monkeys, or donkeys.
  • a reagent used in therapeutic methods of the invention is present in a pharmaceutical composition.
  • Pharmaceutical compositions typically comprise a pharmaceutically acceptable carrier, which meets industry standards for sterility, isotonicity, stability, and non-pyrogenicity and which is nontoxic to the recipient at the dosages and concentrations employed.
  • the particular carrier used depends on the type and concentration of the therapeutic agent in the composition and the intended route of administration.
  • a stabilizing compound can be included. Formulation of pharmaceutical compositions is well known and is described, for example, in U.S. Pat. Nos. 5,580,561 and 5,891,725.
  • a therapeutically effective dose refers to that amount of active ingredient that increases anti-tumor cytolytic T-cell activity relative to that which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination (s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Effective in vivo dosages of polynucleotides and polypeptides are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g.
  • Test substances which can be tested for use as a potential drug or immune enhancing agent can be any substance known in the art.
  • the substance can be previously known for another purpose, or it can be previously unknown for any purpose.
  • the substance can be a purified compound, such as a single protein, nucleic acid, or small molecule, or it can be a mixture, such as an extract from a natural source.
  • the substance can be a natural product, or it can be a synthetic product.
  • the substance can be specifically and purposefully synthesized for this purpose or it can be a substance in a library of compounds which can be screened.
  • One type of antibody response was that against a galactoside-binding protein galectin-3. Eight out of 12 patients with Disease Free Survival (DFS)>3 years developed galectin-3-specific antibody response as compared with patients with DFS ⁇ 3 years in which only 2 out of 21 patients did. This type of antibody response is characterized by a close relationship between antibody response development and vaccination, indicating that the antibody response was induced and/or augmented by the vaccine. The fact that galectin-3-specific antibody response in 4 patients was transiently suppressed by radiation and chemotherapy implies a recently developed response possibly by 1 s ′ vaccination.
  • DFS Disease Free Survival
  • Galectin-3 is a member of the ⁇ -galactoside-binding lectin (galectin) family which contains a conserved C-terminal carbohydrate-recognition-binding domain and a unique N-terminal proline- and glycine-rich domain (36). It is ubiquitously expressed in different types of cells and tissues and has multiple biological functions depending on its subcellular localization. Galectin-3 is mainly a cytoplasmic protein, but can easily translocate into the nucleus or be secreted to the extracellular milieu.
  • Nuclear galectin-3 is involved in pre-mRNA processing, cell cycle regulation, and regulation of cancer-related gene expression (37). Cytoplasmic galectin-3 has anti-apoptotic activity by interacting with several apoptosis regulators such as Bcl-2, CD95, nucling, and Alix/AIP 1. It also modulates several signaling pathways including K-Ras signaling. Extracellular galectin-3 mediates cell adhesion, migration, and cell-cell interactions (36). Expression of galectin-3 in a variety of tumors has been associated with high invasiveness, tumor progress, and metastasis (38-40).
  • galectin-3 is also a direct negative regulator of T cell activation by interfering TCR-CD8 colocalization in CD8 T cell activation (41) or downregulating TCR and thus destabilizing immunological synapse in CD4 T cell activation (42).
  • Small molecule inhibitors targeting galectin-3 have been shown to counteract its anti-apoptotic activity and enhance chemosensitivity and radiosensitivity of cancer cells. When combined with chemotherapy, these inhibitors significantly reduce cancer metastasis and increase survival in an animal model (43, 44).
  • galectin-3 antibody response was associated with a favorable clinical outcome could implicate a new line of cancer immunotherapeutic agent, i.e., anti-galectin-3 antibody. Whether anti-galectin-3 antibody acts as a neutralizer to reverse galectin-3's negative regulation of T cell activation or acts on its other cancer-related functions remains to be determined
  • Antibody response to AnnexinA2, enolase ⁇ and RhoGDI ⁇ could be detected at the very early pre-malignant stage of cancer development in an animal model (4), which implies that antibody response to these proteins in patients of this study might have existed long before cancer was diagnosed but was suppressed at the time of diagnosis and recovered after radiation and chemotherapy.
  • tumor growth and tumor immune response co-exist, which represents an extremely complex interaction between pro-tumor and anti-tumor factors; such factors involve not only diverse elements of innate and adaptive immunity but also tumor per se and its microenvironment.
  • an immune suppressive tumor microenvironment When a tumor progresses to a stage that can be clinically detected, an immune suppressive tumor microenvironment usually develops which includes immunosuppressive tumor-associated macrophages, myeloid-derived suppressor cells, regulatory T cells, and tumor-derived immunosuppressive products (such as VEGF, TGF-(3 and IL-10) (57). Radiotherapy and chemotherapy could possibly reverse this immunosuppressive tumor microenvironment by eliminating myeloid-derived suppressor (58), regulatory T cells (59), and tumor-derived suppressive factors (60), thus augmenting the pre-existing immune response.
  • immunosuppressive tumor-associated macrophages such as myeloid-derived suppressor cells, regulatory T cells, and tumor-derived immunosuppressive products (such as VEGF, TGF-(3 and IL-10) (57).
  • Radiotherapy and chemotherapy could possibly reverse this immunosuppressive tumor microenvironment by eliminating myeloid-derived suppressor (58), regulatory T cells (59), and tumor-derived suppressive factors (60), thus augmenting
  • Cell surface membrane proteins are the ideal targets for antibodies. Due to their high hydrophobicity, membrane proteins tend to precipitate at their isoelectric point during isoelectric focusing so that they are difficult to be resolved on a conventional two-dimensional gel electrophoresis used in this study. All the proteins identified in this study are traditionally classified as intracellular proteins. However, although lacking a secretion signal sequence, many proteins can translocate to cell membrane via non-classical pathway under certain patho-biological conditions including cancer where they bind to their partners and exert different functions. In this sense, these “ectopic” proteins could be the more suitable immune targets because they may be absent at the surface of normal cells. As mentioned above, galectin-3 has a membrane form that mediates cell adhesion, migration, and cell-cell interactions (36).
  • Cell surface AnnexinA2 serves as receptor for both tenascin C and tissue plasminogen activator (tPA) which promote tumor angiogenesis and progression (45, 49).
  • tPA tissue plasminogen activator
  • Post-translational phosphorylation of AnnexinA2 in pancreatic cancer cells is required for its membrane localization and cell invasion.
  • enolase ⁇ recruits plasminogen to the cell surface so that fibrinolysis takes place in the vicinity of the cell (50).
  • Such a pericellular fibrinolytic activity facilitates cell's mobility and invasiveness. Indeed, enolase ⁇ has been associated with increased venous invasion of hepatocellular carcinoma (51). Increased amount of HSP60 on the cell surface has been seen as “danger signal” for the immune system.
  • RhoGDI ⁇ is a regulator of Rho GTPases which are involved in a variety of cell signaling pathways (54). RhoGDI ⁇ can protect cancer cells from apoptosis induced by chemotherapeutic agents (55) and its overexpression was associated with tumor progression and poor prognosis in colorectal cancer (56).
  • Antibody response to certain tumor associated antigens was reported to be correlated with tumor burden, thus predicting poor prognosis (14-17).
  • Shebzukhov, et al. reported that antibody response to a SEREX-defined colon cancer TAA, thymidylate synthase, was detected in colon cancer patients only after 5-FU-based chemotherapy and the antibody titer was associated with tumor burden (61).
  • the radio-chemotherapy-enhanced antibody response we observed in this study was clearly associated with a favorable prognosis ( FIG. 5 ).
  • the correlation between antibody titer and tumor burden, if any, was inverse as enolase ⁇ specific antibody titer dropped drastically over time in a patient with an early disease relapse ( FIG. 5B ).
  • AnnexinA2 in mediating PDAC invasion and metastases.
  • the translocation of AnnexinA2 from the cytosol/enodsome compartment to the cell membrane is required for AnnexinA2 mediated PDAC cell invasion.
  • phosphorylation at tyrosine 23 is critical for this translocation to occur in PDAC cells.
  • AnnexinA2 translocation in PDAC is mediated by TGF-beta.
  • translocation of AnnexinA2 in PDAC is associated with EMT in these cells, further confirming that AnnexinA2 is important to the mechanism by which PDAC cells progress and metastasize.
  • AnnexinA2 was brought to our attention when we employed the serum from vaccinated patients to screen a panel of tumor antigens targeted by vaccine induced humoral immune responses. Additional studies are underway to determine whether the induction and maintenance of AnnexinA2 humoral responses correlates with improved clinical responses. However, in this study, we focused on characterizing the role of AnnexinA2 in PDAC invasion.
  • AnnexinA2 is overexpressed in PDAC in comparison with paracancerous normal pancreatic ductal epithelium (Esposito, Penzel et al. 2006).
  • the cell surface/membrane localization of AnnexinA2 may be a more specific marker for PDAC since this cell surface localized fraction of AnnexinA2 appears to correlate with PDAC pathogenesis.
  • Cell surface AnnexinA2 starts to increase in the PanINs and further increases when the PanINs develop into invasive PDAC.
  • AnnexinA2 or the AnnexinA2/S100A10 heterotetramer have also been shown to be a high-affinity receptors for multiple extracellular ligands such as tissue plasminogen activator (tPA), plasmin, plasminogen, progastrin/gastrin, tenascin-C, and angiostatin, and all are hypothesized to be mediators of cancer cell invasion and metastases (Kim and Hajjar 2002; Kwon, MacLeod et al. 2005; Sharma and Sharma 2007).
  • tPA tissue plasminogen activator
  • AnnexinA2 Different subcellular localizations, including membrane, cytoplasmic, and nuclear localizations, have all been reported for AnnexinA2 (Kim and Hajjar 2002; Rescher and Gerke 2004; Sharma and Sharma 2007; Singh 2007). AnnexinA2 is also secreted extracellularly (Lu, Maeda et al. 2006). The presence of AnnexinA2 in different subcellular fractions is consistent with its multiple functions. It is also likely that AnnexinA2 localizes to different locations in different cell types or under different conditions (Liu, Rothermund et al. 2003; Deora, Kreitzer et al. 2004). AnnexinA2 is sometimes localizes to the cell surface in normal pancreatic ductal epithelium.
  • AnnexinA2 appears to be well organized along the apical surface of the pancreatic ductal epithelium. This is in line with the polarized expression of AnnexinA2 in many normal tissue types (Massey-Harroche, Mayran et al. 1998) and supports the requirement of AnnexinA2 for the formation of the apical surface and lumen in the three-dimensional Madin-Darby canine kidney cell system, which is a model of kidney development. By contrast, the polarized expression of AnnexinA2 is disrupted in PanINs and PDAC, even though the lumen structure is still maintained in these lesions.
  • AnnexinA2 translocation to the cell surface/membrane in PDAC is mediated by TGF-beta.
  • TGF-beta the translocation of AnnexinA2 in PDAC is associated with EMT in these cells as characterized by the downregulation of epithelial cell markers and the upregulation of mesenchymal cell markers.
  • AnnexinA2 has previously been shown to mediate TGF ⁇ -activated EMT in cardiac valve development during embryogenesis (Krishnan, Deora et al. 2004).
  • the EMT is a highly conserved normal cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells during organ development.
  • E-cadherin an epithelial marker
  • N-cadherin a mesenchymal marker, induced in PDAC
  • the results described below support AnnexinA2 as a biomarker of and immunogenic protein expressed by PDAC. Moreover, the results below support a role for cell surface AnnexinA2 in PDAC invasion, and supports the development of AnnexinA2 as a novel PDAC therapeutic target.
  • FIG. 1 shows that each patient had a different but distinct antibody response profile in which antibody activities were seen against a fix set of proteins of different sizes for all the time points with one or two dominant responses.
  • a few antibodies showed up only in the post-vaccination sera but, more often, titer of antibodies changed in the post-vaccination or post-radio-chemotherapy sera.
  • Antibody Response Correlates with Favorable Clinical Response
  • Resulting peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry.
  • MALDI-TOF matrix-assisted laser desorption ionization time-of-flight
  • Galectin-3-Specific Antibody Response Correlates with Vaccinations and Clinical Outcome
  • Antibody titer started to rise just after 1 St vaccination in 4 of the 8 patients in DFS>3 yr group who had an elevated antibody response in post-vaccination sera, but dropped slightly after radiation and chemotherapy, and after 2 nd vaccination the antibody titer resumed to the post-1 st vaccination level and peaked after 4 th vaccination ( FIG. 4C in solid symbols and solid lines).
  • the other 3 patients in the DFS>3 yr group ( FIG. 4C in open symbols and dashed lines) showed a delayed galectin-3 antibody response which occurred after 3 vaccinations.
  • Patient 6 from DFS ⁇ 3 yr group also had a high pre-existing enolase ⁇ antibody titer, but the titer dropped drastically over time to the lowest level after 3 rd vaccination when disease relapsed.
  • Patient 9 had medium level (in relative to enolase ⁇ antibody in patient 10) of pre-existing antibodies to AnnexinA2 and HSP60 but negative for RhoGDI ⁇ antibody in pre-vaccination serum.
  • Antibody titers in patient 9 to all 3 proteins increased more than 2-fold post radiochemotherapy and kept relatively stable up to post 4 th vaccination (the last vaccination for patient 9).
  • Antibody titers to AnnexinA2 and HSP60 also increased dramatically post radiochemotherapy in patient 47, declined gradually afterwards and returned to the pre-vaccination level after 5 th vaccination.
  • Pancreatic cancer cell lines PANC 10.05 and PANC 6.03 were derived from two histologically confirmed primary pancreatic adenocarcinomas.
  • Vaccine cell lines used in the clinical trial were developed by genetically modifying these two cell lines to secrete human GM-CSF (62).
  • GM-CSF human GM-CSF
  • the unmodified parental cell lines were used in the present study as a source of antigens for testing immune response.
  • the cell lines were cultured as described previously (62).
  • Total cell lysate was prepared by lysing PANC 10.05 and PANC 6.03 cells in lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM sodium chloride, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulfate, and 0.02% soudium azide) supplemented with protease inhibitor cocktail (Sigma) and 1 mM PMSF. Protein concentration was determined by BCA assay (Pierce Biotech). Proteins in the lysate were resolved at 40 ⁇ g/lane on Criterion XT 4-12% Bis-Tris precast gels (Bio-Rad) in MOPS running buffer.
  • Cell lysate for 2-DE was prepared by lysing PANC 10.05 and PANC 6.03 cells in isoelectric focusing (IEF) buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, and 0.5% carrier ampholytes, pH3-10). Protein concentration was estimated by Bradford assay. Cell lysate was pre-fractionated on a ZOOM IEF Fractionator (Invitrogen) into 2 fractions which contained proteins with isoelectric points from pH 3-6 and from pH 7-10, respectively by following manufacturer's instruction manual.
  • IEF isoelectric focusing
  • Protein spots on the Coomassie-stained gels corresponding to the antibody-reactive proteins revealed on the images of Western blot analysis were excised. Trypsin in-gel digestion was performed using Trypsin Profile IGD Kit (Sigma) as instructed in manufacturer's manual. The resulting peptide mixture was cleaned up with a ZipTip C 18 pipette tip (Millipore) and eluted directly onto a MALDI sample plate in 2 ⁇ l of MALDI matrix of 10 mg/ml of ⁇ -cyano-4-hydroxycinnamic acid in 70% acetonitrile with 0.1% trifluoroacetic acid.
  • MALDI-TOF MS analysis was performed in reflection positive ion mode on a Voyager-DE STR System (Applied Biosystems). Protein identity was obtained by searching the monoisotopic masses against the NCBInr database at a tolerance of 100 ppm using Mascot Peptide Mass Fingerprint online search engine (www.matrixscience.com).
  • NPM1 cDNA was amplified by PCR from plasmid pNPM1NS1 (kindly provided by Dr. Alexander M. Shneider) which contains viral NP and M1 sequences from influenza strain A/WSN/33-H1N1 (63).
  • a fusion gene with an 8 ⁇ His tag sequence at the 3′-terminus was inserted into a mammalian expression vector pcDNA3.3-TOPO (Invitrogen).
  • the 8 ⁇ His tagged recombinant protein was transiently expressed in 293 T cells by transfecting the cells with the protein-coding vector by Lipofectamine 2000-mediated method (Invitrogen) and purified with His GraviTrap Prepacked Ni Sepharose affinity column (GE Healthcare) per manufacturer's instruction. The purity of the protein was confirmed by SDS-PAGE analysis, Western blotting with a few selected serum samples, and MALDI-TOF MS.
  • Costar 3690 96-well half-area EIA/RIA plates (Corning) were coated with 30 ⁇ l/well of purified recombinant proteins at 5 ⁇ g/ml (galectin-3, HSP60, RhoGDI ⁇ , and NPM1) or 10 ⁇ g/ml (AnnexinA2 and enolase ⁇ ) in bicarbonate/carbonate coating buffer at 4° C. overnight.
  • the protein-coated plates were incubated with 150 ⁇ l/well of ELISA Blocker Blocking Buffer (Pierce Biotech) for 1 h at room temperature.
  • the wells were then incubated with 30 ⁇ l/well of serial dilutions (1:100, 1:200, 1:400, and 1:800) of sera (duplicates for each dilution) for 2 h at room temperature and with 30 ⁇ l/well of 1:200,000 dilution of goat anti-human IgG ( ⁇ -chain specific) peroxidase conjugate (Sigma, A8419) for 1 h at room temperature.
  • the wells were washed extensively with TBS-T between incubations.
  • 30 ⁇ l/well of ready-to-use 3,3′5,5′-tetramethylbenzidine (TMB) liquid substrate (Sigma, T0440) was added to the wells and incubated in dark for 20 min at room temperature.
  • TMB 3,3′5,5′-tetramethylbenzidine
  • the color development was stopped by adding to the wells 30 ⁇ l/well of 1 N sulfuric acid. Absorbance at 450 nm (with a reference wavelength of 570 nm) was measured on a PowerWave 340 microplate reader (BioTek). For each protein, an ELISA optimization was performed in advance with a positive serum sample (based on Western blot analysis) to have an OD 450 of about 1.000 at serum dilution of 1:100. For each experiment, a control ELISA was performed simultaneously with a second set of plates coated with only coating buffer for background subtraction. Antibody titer in a serum sample was reported as OD 450 at serum dilution of 1:400 after background subtraction.
  • AnnexinA2 as a Potential Tumor Associated Antigen and Biomarker of PDAC
  • AnnexinA2 as a new candidate tumor antigen that may be involved in PDAC development and progression. Specifically, we show that cell cytoplasmic to cell surface/membrane translocation of AnnexinA2 occurs with PDAC development and progression, and occurs as a result of the phosphorylation of tyrosine 23 on AnnexinA2. We also show that the translocation of AnnexinA2 plays an important role in PDAC invasion and that loss of AnnexinA2 translocation to the cell membrane leads to loss of TGF ⁇ -induced EMT in pancreatic cancer cells.
  • AnnexinA2 is one protein that was found to be recognized by post-vaccination sera from both patients evaluated.
  • purified recombinant AnnexinA2 from mammalian cells was first produced and confirmed to be pure by Coumassie blue stain ( FIG. 7A ), and then used to screen pre-vaccination and post-vaccination serum from 16 patients treated in this phase II clinical trial by western blot ( FIG. 7B ).
  • AnnexinA2 is reported to be overexpressed in a variety of cancers including PDAC when compared with normal tissues (Vishwanatha, Chiang et al. 1993; Esposito, Penzel et al. 2006).
  • Normal pancreatic ductal epithelial cells usually show weak cytoplasmic and lumenal staining of AnnexinA2 by immunohistochemistry (IHC) analysis.
  • IHC immunohistochemistry
  • AnnexinA2 Mediates the Invasion of Pancreatic Cancer Cells
  • AnnexinA2 mainly binds membrane associated phospholipids and cytoskeleton, and is also associated with the extra-cellular surface of cells, functioning as a high-affinity receptor for multiple ligands such as tissue plasminogen activator (tPA), plasmin, plasminogen, progastrin/gastrin, tenascin-C, and angiostatin.
  • tissue plasminogen activator tPA
  • plasmin plasminogen activator
  • progastrin/gastrin tenascin-C
  • angiostatin angiostatin
  • AnnexinA2 is a regulatory mechanism that confers PDAC cells with invasion capacity.
  • pancreatic cancer cells vary in their invasion capacity ( FIG. 9A ).
  • 8 of these cell lines have higher invasion capacity and 3 have lower invasion capacity when compared with a normal fibroblast cell line.
  • proliferation rates of selected cell lines and did not appreciate any correlation between their proliferation rate and invasion capacity (data not shown).
  • AnnexinA2 is slightly lower in cells with lower invasion capacity and slightly higher in those with higher invasion capacity, suggesting that over expression of AnnexinA2 in PDAC may contribute to the pancreatic cancer cell's greater invasion potential. Nonetheless, expression levels of AnnexinA2 in whole cell extracts vary to a much less extent than the invasion capacity, suggesting that other regulatory mechanisms play a dominant role in determining the invasion capacity of PDAC cells.
  • AnnexinA2 In an attempt to uncover other regulatory mechanisms that account for the invasion capacity of PDAC cells, we examined the subcellular localization of AnnexinA2 in various pancreatic cancers by fluorescent staining with anti-AnnexinA2 antibodies. The immunostaining of AnnexinA2 in representative cells with higher or lower invasion capacity is shown in FIG. 9C . Interestingly, as shown in Table 3, AnnexinA2 is predominantly localized to the cell membrane in all 8 PDAC cell lines tested with higher invasion capacity. In contrast, AnnexinA2 is found localized to the cytoplasm and/or nucleus in the 2 out of 3 pancreatic cancer cells tested with lower invasion capacity and in the non-cancerous fibroblast cell line.
  • AnnexinA2 is phosphorylated at Tyrosine 23 (Tyr23) when it is localized to the cell surface under stress (Deora, Kreitzer et al. 2004). Malignant cells often mimic normal cells that have been subjected to a variety of stress stimuli. We therefore hypothesized that AnnexinA2, when localized to the cell surface of PDAC cells is also a tyrosine phosphoprotein. To test our hypothesis, we eluted the cell surface fraction of AnnexinA2 from the Panc10.05 PDAC cells which have a high invasion capacity. We found that the cell surface fraction of the AnnexinA2 protein is in fact a tyrosine phosphorylated protein, when detected using the anti-phosphotyrosine antibody ( FIG. 10A ). In contrast, AnnexinA2 is not eluted from the cell surface of Panc 3.11 cells, which is one of the PDAC cell lines that demonstrated lower invasion capacity.
  • AnnexinA2 is a major substrate for the Src kinase, and this kinase phosphorylates AnnexinA2 in vivo at the Tyr23 residue (Sharma and Sharma 2007).
  • LV-ANXA2WT exogenous AnnexinA2
  • Y23A point mutation
  • Y23E point mutation
  • AnnexinA2 localized to the cell surface in uninfected Panc10.05 cells and Panc10.05 cells infected by the lentiviruses expressing either ANXA2WT or the mutant ANXA2Y23E.
  • AnnexinA2 localized to the cytoplasm in Panc10.05 cells infected with the lentivirus expressing the mutant protein, ANXA2Y23A.
  • immunostaining of AnnexinA2 detected both exogenous and endogenous AnnexinA2 at the same time. Even the endogenous AnnexinA2 no longer localized to the cell surface in the cells infected with LV-ANXA2Y23A, suggesting that ANXA2Y23A had a dominant negative effect.
  • Panc10.05 cells transfected with the plasmid expressing ANXA2Y23E-GFP localized to the cell surface demonstrate that phosphorylation at Try23 is critical for the localization of AnnexinA2 to the cell surface.
  • AnnexinA2 Try23 phosphorylation of AnnexinA2 affects the localization of AnnexinA2.
  • the expressed exogenous AnnexinA2 is resistant to RNA interference because of mutations within the siRNA target site when these plasmids are cotransfected with the AnnexinA2 siRNA.
  • ANXA2 WT-FLAG becomes tyrosine phosphorylated in the cell membrane fraction.
  • siRNA duplex that specifically targets only endogenous AnnexinA2.
  • transfection with the empty pcDNA vector had no effect on the in vitro invasion of Panel 0.05 cells.
  • transfection with either ANXA2 WT-FLAG or ANXA2Y23E-FLAG had a dominant negative effect on the invasion of Panel 0.05 cells.
  • transfection with ANXA2Y23A-FLAG further inhibited the invasion of Panel 0.05 cells, and co-transfection of the empty vector with the AnnexinA2 targeting siRNA inhibited invasion to the same extent.
  • AnnexinA2 Plays a Role in the Epithelial-Mesenchymal Transition
  • the initial step of invasion-metastasis mimics Epithelial-Mesenchymal Transition (EMT), a normal morphogenic process during embryonic development (Weinberg 2008).
  • EMT Epithelial-Mesenchymal Transition
  • AnnexinA2 has been shown to mediate TGF ⁇ -activated EMT during the process of cardiac valve development (Krishnan, Deora et al. 2004). It has been repeatedly shown that TGF ⁇ can induce EMT in cultured PDAC cells (Gordon, Dong et al. 2008; Zhao, Venkatasubbarao et al. 2008).
  • previous studies have demonstrated that EMT mediates invasion and metastases of PDAC (Zhao, Venkatasubbarao et al. 2008).
  • AnnexinA2 may play a key role in mediating the EMT process during PDAC invasion and metastasis.
  • EMT is characterized by a typical transcription circuit of events. The transcription of epithelial markers such as E-cadherin are suppressed and that of mesenchymal markers such as slug and vimentin are induced.
  • a lentiviral vector containing AnnexinA2 siRNA as a method to achieve long-term suppression of AnnexinA2.
  • Panc10.05 cells infected with this lentivirus were FACS-sorted by GFP, which was co-expressed by the lentivirus.
  • the human pancreatic cancer cell lines except MiaPaca-2 were previously established by the Johns Hopkins pancreatic tumor GI SPORE research program (Jones, Zhang et al. 2008).
  • MiaPaca-2 was originally obtained from the American Type Culture Collection.
  • the human fibroblast cell line was established from paracancerous tissues of human pancreatic adenocarcinoma. All cell lines were maintained in the RPM11640 media supplemented with 10% fetal bovine serum and grown in a humidified incubator at 37 oC and 5% CO 2 .
  • TGF ⁇ 1 R&D Systems
  • Human serum was obtained from the patients enrolled in the phase II pancreatic vaccine adjuvant study by following the IRB-approved protocol (Lutz et al. Manuscript submitted). Serum was collected and stored according to standard procedures (Jaffee, Hruban et al. 2001). Rabbit polyclonal anti-AnnexinA2 antibodies (HSO) were obtained from Santa Cruz Biotechnology, Inc.
  • the full-length human AnnexinA2 cDNA was obtained by reverse transcription of total RNA purified from Panc10.05 cells, followed by high-fidelity PCR amplification with the AnnexinA2 primers.
  • the non-complementary region of the reverse primer also contained the sequence of FLAG tag.
  • the resultant PCR product of the AnnexinA2 cDNA was then cloned into the pCR vector (Invitrogen) and was sequenced to confirm no introduction of missense or nonsense mutations.
  • the AnnexinA2 cDNA fragment with a C-terminal FLAG tag was further subcloned into the lentiviral vector (LV).
  • AnnexinA2 is expressed under the control of the EF-1 ⁇ promoter.
  • the resultant PCR product of the AnnexinA2 cDNA with a C-terminal FLAG tag was cloned into the pcDNA3.3 vector (Invitrogen) directly.
  • Y23A and Y23E mutations were created by the site-directed mutagenesis according to the manufacturer's manual (Stratagene).
  • plasmid transfection and RNA interference cells were seeded in multiple 6-well plates to 80% confluence. For each well, 2 ⁇ g of pcDNA-based plasmid and/or 40 pmol siRNA duplex, were transfection with the lipofectamine 2000 reagent in a serum-containing medium according to the manufacturer's manual (Invitrogen). For protein expression analysis, cells were harvested in 48 hours. For in vitro invasion assay, cells were starved in serum-free media for another 24 hours. The AnnexinA2 siRNA was synthesized by Ambien, Inc.; and the scramble siRNA was also purchased from Ambion.
  • the plasmid with lentiviral constructs was co-transfected with packaging plasmids into 293T cells as previously described (Zhou, Cui et al. 2003). Lentivirus supernatant was collected at 48 hours. For infection, cells were seeded in multiple 6-well plates to 80% confluence. For each well, 2 ml lentivirus supernatant was added and incubated for 48 hours before the cells were harvested.
  • the lentivirus expressing hairpin siRNA of AnnexinA2 was obtained from Open Biosystems. Lentivirus was produced according to the manufacturer's manual. For infection of Panc10.05 cells, 6 milliliters of viral supernatant was added to adherent cells plated in each 75 cm flask and incubated for 48 hours. Cells from two flasks were sorted by GFP in a FACS cell sorter at approximately 72-96 hours after infection. The cells infected with lentivirus expressing GFP alone were sorted similarly. Total RNA was immediately extracted after cell sorting.
  • invasion specificity controls were also performed by only adding serum-free media in the bottom wells.
  • relative MTT units in the invasion experiments are adjusted by subtracting the MTT values of leaked cells in matched invasion specificity controls.
  • Panc10.05 cells grew on cover slips to 90% confluence and were fixed in 4% paraformaldehyde for 15 min. Cover slips were then incubated with PBS containing 0.1% Triton X-100 for 5 minutes followed by washing with PBS. After cover slips were blocked with 10% normal goat sera in PBS for one hour, they were incubated with rabbit anti-AnnexinA2 antibodies at a 1:100 dilution in 10% normal goat sera overnight at 4 oC. Following a PBS wash, they were further incubated with FITC-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) at a 1:200 dilution in 10% normal goat sera at room temperature for 1 hour.
  • cover slips were mounted in a medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector Labs) and examined by a fluorescent microscope.
  • Each area of PDAC cells on the entire slide will be scored from 0 to 3 by clinical pathologists. Scores of 0 to 3 measure the different intensities of cell-surface staining of AnnexinA2, with a score of 0 representing no staining and a score of 3 representing the strongest staining. The percentage of PDAC cells at each score level will be estimated. The average score of cell-surface AnnexinA2 expression is calculated as follows:
  • pancreatic cancer cells The whole cell extract of pancreatic cancer cells was obtained as previously described (Chen, Riley et al. 1997). In brief, cell pellets were resuspended in the Lysis 250 buffer followed by a freeze and thaw process that was performed three times. The cell lysate was spun at 15,000 rpm for 10 min and the supernatent was removed. The protocols to separate membrane and cytoplasmic fractions were adapted from those previously published (Abrams, Rohrschneider et al. 1982).
  • EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid
  • Anti-phosphotyrosine antibody conjugated sepharose (P-Try-100, Cell Signaling Technology) was used to immunprecipate tyrosine-phosphorylated proteins.
  • Anti-AnnexinA2 antibodies and anti-FLAG M2 antibodies (Sigma) were first conjugated to sepharose beads according to the manufacturer's manual (Pierce) prior to being used for immunoprecipation. All immunoprecipations were done at 40 C for overnight, followed by washing with the Lysis 250 buffer (Chen, Riley et al. 1997).
  • Recombinant His6-tagged AnnexinA2 was expressed in TOP10 E. coli and purified on a High-Trap Ni column according to the manufacturer's manual (Amershan Pharmacia). One microgram of purified His6-tagged AnnexinA2 was loaded on each well of a 10% gradient SDS-PAGE. After transferring to the membrane, each individual lane was blotted with either pre-vaccination serum or post-vaccination serum at a 1:1000 dilution. Mouse anti-human IgG antibody (Sigma) was used as a 1:5000 dilution as the secondary antibody.
  • qRT-PCR Quantitative real-time reverse transcription-PCR

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